Composition and method to enhance the efficacy of a fish vaccine and to stimulate the immune system of fish

ABSTRACT

A method to stimulate the immune system of fish that comprises administering to the fish a β-1,3-glucan having a β-1,3-linked main chain with β-1,6-linked single glucose side chains.

This application is a continuation-in-part application of theapplication Ser. No. 07/313,033 having a filing date of Feb. 21, 1989,now U.S. Pat. No. 5,147,862.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method to enhance the efficacy of a fishvaccine and to a method to stimulate the immune system of a fish and toa composition and an agent useful in these methods.

2. Discussion of Background

Fish rearing in fish farms is increasingly evident. The advantages ofaquaculture in comparison to fishing wild stocks is as apparent as thekeeping of cattle compared with the hunting of wild animals. However,there are problems. The fish must be kept at a very high density andthis means that the fish are susceptible to disease and, in particular,to the rapid spread of disease through the fish farm. Aquaculturetechniques are improving all the time and the use of antibiotics,usually administered in the food, has been a major advantage in reducingdisease. Nevertheless the use of antibiotics in this way can lead to thedevelopment of resistant strains of fish pathogens. There is also apopular sentiment against the use of antibiotics, growth hormones andthe like in livestock and fish to be consumed by humans.

There has been some success in the use of vaccines, notably againstdiseases such as vibriosis and enteric redmouth. But there are stilldiseases for which vaccines are not effective or are of inadequateefficacy.

It is known in medicine that the efficacy of certain vaccines can beimproved by the administration of compounds in addition to the vaccines.

It is known that certain glucans can be useful in curing human cancer byan apparent immunomodulating effect. Glucans are the anhydrides ofglucose derivatives, for example cellulose, starch, dextrin andglycogen.

Relevant patents relating to glucans known to applicant include U.S.Pat. Nos. 4,098,661 issued Jul. 4, 1978, British Patent 1,061,043 datedOct. 23, 1963 and Canadian Patent 968,286 issued May 27, 1975.

It has now been observed that certain glucans exhibit appreciable,advantageous effect in the use of vaccines for treating fish diseases.Of particular interest are β-1,3-glucans and particularly the compoundsschizophyllan (SPG) and scleroglucan (SG). These water solubleβ-1,3-glucans are reported to have triple helical structures.Schizophyllan is prepared by the precipitation in a culture filtrate ofthe fungi Schizophyllum commune, using acetone, ethanol or other watermiscible solvent as a precipitant. Scleroglucan is obtained in a similarmanner from a culture filtrate of a selected species of Sclerotium. Theabove compounds are represented by the following structural formulae:##STR1##

SUMMARY OF THE INVENTION

The present invention is thus directed to the immunostimulating andvaccine enhancing effects of β-1,3-glucans and is based on thesurprising observation that these compounds are effective in thetreatment of fish and in vaccine prophylaxis in fish.

Accordingly, in a first aspect, the present invention is a method tostimulate the efficacy of a fish vaccine that comprises administering toa fish treated with the vaccine a β-1,3-glucan having a β-1,3-linkedmain chain with β-1,6-linked single glucose side chains.

In a second aspect the invention is a method to stimulate the immunesystem of fish that comprises administering to the fish a β-1,3-glucanas defined above.

In yet a further aspect the invention is a composition able to enhancethe efficacy of a fish vaccine and comprising a β-1,3-glucan havingβ-1,3-linked main chain with β-1,6-linked single glucose side chainswith an antigen/β-1,3-glucan weight ratio of from 0.015 to 0.15.Preferably the antigen source is a vaccine.

In yet a further aspect the invention is an agent to administer to afish to stimulate the immune system of the fish comprising an effectiveamount of β-1,3-glucan having a β-1,3-linked main chain withβ-1,6-linked single glucose side chains and carrier acceptable to thefish.

The carrier will typically be saline for injectable agents and food foragents to be administered by mouth. With advantage alpha-cellulose maybe mixed with the glucan prior to mixing with food.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 relates cumulative mortality to time in certain comparativeexperiments described in the Experimental Work below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Experimental Work

Experimental procedures were developed to determined the ability ofcertain β-1,3-glucans to enhance the performance of formalin killedbacterin and stimulate the non-specific immune system of salmonid fishessuch that they can show increased survival during a virulent challenge.The model system chosen for the challenge was Aeromonas salmonicida theetiological agent of furunculosis, because of the reported involvementof cell mediated immunity in this disease. Cell mediated immunity playsa major role in immune protection against bacterial kidney disease(BKD), another disease that plagues aquaculture fish, but the course ofinfection is long, necessitating the use of a furunculosis model system.

EXAMPLE 1

Two systems of challenge were used. First immersion, secondlycohabitation. The mortality figures were then adjusted by calculatingrelative potency so that the comparisons could be made between the twochallenge groups. Relative potency showed good reproducibility betweentanks and lentinan and schizophyllan enhanced vaccines were both foundto have similar level of potency within and between tanks. The relativepotency was determined according to Table 1. Relative potency is definedas percentage mortality in the control divided by the percentagemortality in the vaccinates.

The results are set out in Table 1.

                  TABLE 1                                                         ______________________________________                                        Enhancement of efficacy of                                                    injection-delivered vaccine by the β-1,3-Glucans,                        Lentinan and Schizophyllan (SPG).                                             Challenge                         *Relative                                   Method      Treatment    % Mort.  Potency                                     ______________________________________                                        Immersion   Lentinan + Ag                                                                              60%      1.6                                                     SPG + Ag     64%      1.5                                                     Antigen only 96%      1.0                                         Cohabitation                                                                              Lentinan + Ag                                                                              52%      1.6                                                     SPG + Ag     52%      1.6                                                     Antigen only 84%      1.0                                         ______________________________________                                         Ag: Antigen                                                                   *Relative Potency is defined as follows:                                      ##STR2##                                                                 

Because of the similarity in structure function the followingexperimental work was conducted with one compound, schizophyllan. Thiscompound is less costly to produce than lentinan. It can be produced bybatch fermentation but lentinan must be extracted from the fruiting bodyof the mushroom Lentinus edodes, which is a popular food in Japan andelsewhere, making the compound lentinan expensive.

EXAMPLE 2

In the next phase of experiments SPG was obtained in three differentforms. First a purified and extensively depolymerized form (SPG-P).Secondly a crude extract with only slight depolymerization (SPG-C) and,thirdly, a crude preparation of native SPG in culture broth containingpulverized mycelia (SPG-M).

These forms of SPG were administered to fish by intraperitoneal (i.p.)injection in an admixture containing A. salmonicida bacterin and oneeach of the three different forms of SPG. Fish were then challenged byimmersion in virulent A. salmonicida and the resulting mortalities weremonitored. The results are in Table 2.

                  TABLE 2                                                         ______________________________________                                        Differences in Efficacy of various molecular                                  weight forms of SPG.                                                          Treatment % mortality   % morality                                                                              pooled                                      received  replicate 1   replicate 2                                                                             % mort.                                     ______________________________________                                        SPG-M1 + Ag                                                                              6.7%         26.7%     16.7%                                       SPG-C2 + Ag                                                                             60.0%          3.3%     31.7%                                       SPG-P3 + Ag                                                                             56.7%         66.7%     61.7%                                       Ag only   33.3%         20.0%     26.7%                                       saline    76.7%         66.6%     71.7%                                       ______________________________________                                         1 SPGM designates the crudest form of SPG. This is a preparation of           colloidaly pulverized mycelia containing the highest molecular weight of      SPG.                                                                          2 SPGC is a crude form of SPG that has been somewhat purified and somewha     depolymerized.                                                                3 SPGP is the purified and depolymerized form of SPG.                    

It is seen from the above that the crudest form of SPG (SPG-M) was themost effective. With a decrease in molecular weight there was a declinein the level of enhancement to the vaccine.

EXAMPLE 3

A further series of experiments was conducted to assess the efficacy ofSPG as a non-specific chemoprophylactant. It was found that wheninjected i.p. at the rate of 20 mg/kg 20 days prior to challenge, fishwere protected for at least 40 days after injection to a similar extentto fish that received an i.p. delivered vaccine consisting of antigenalone. These results are summarized in FIG. 1, which graphically relatescumulative percentage mortality to time. The curve is a mortality curveshowing saline-injected fish compared with fish injected with SPG aloneand with antigen alone. Fish injected with SPG alone show a similarlevel of protection to those vaccinated with antigen only. Thisprotection seems to last about 20 days after challenge. The fish wereinjected with the SPG and the antigen 20 days before day zero of thecurve of FIG. 1.

Experimental procedures were developed to determine the ability ofcertain β-1,3-glucans to enhance the performance of formalin killedbacterin and stimulate the non-specific immune system of salmonidfishes.

EXAMPLE 4

A virulent challenge was conducted by using Aeromonas salmonicida theetiological agent of furunculosis by means of immersion. Diet containingcrude schizophyllane (hereinafter referred to simply as SPG-M) andAeromonas salmonicida vaccine (hereinafter referred to simply asvaccine) was orally administered to fish which was attacked by 5.8×10⁵cfu/mL of Aeromonas salmonicida etiological agent, in an amount of 2% ofthe weight for 7 days, and the mortality was examined. The compositionfor the diet is shown in Table 3 and mortalities relative to the ratioof SPG-M to vaccine are shown in Table 4. The diet for control containsno SPG-P or vaccine. Further, data for single use of vaccine are alsoshown in Table 4.

It is evident from Table 4 that when SPG-M and vaccine are used incombination, the mortality is reduced as compared with the single use ofvaccine even though the dose of the vaccine is reduced to one third. Theratio of antigen to SPG-M is preferably from 0.015 to 0.15.

                  TABLE 3                                                         ______________________________________                                        Composition for diet                                                          Composition         Amount (g/kg diet)                                        ______________________________________                                        Steam Dried Herring Meal                                                                          558.66                                                    Dried Whey           84.6                                                     Blood Flour          48.17                                                    Euphasids (whole frozen)                                                                           21.02                                                    Wheat Middling      126.19                                                    Vitamin Supplement   2.41                                                     Mineral Supplement   7.27                                                     Choline Chloride (60%)                                                                             4.60                                                     Ascorbic Acid        1.84                                                     Permapell            13.81                                                    Herring Oil          91.38                                                    SPG-M + Vaccine      0-20.15                                                  α-Cellulose    40.05-19.90                                              ______________________________________                                    

                                      TABLE 4                                     __________________________________________________________________________    Enhancement of efficacy of orally administered vaccine by SPG                 Contents of SPG and Ag                                                                    Mortality (%)                                                     in diet (g/kg)                                                                            Repricate 1                                                                         Repricate 2                                                                         Repricate 3                                                                         Pooled Mortality (%)                            __________________________________________________________________________    Control     50.0  45.0  62.5  52.5                                            Ag 0.45     37.5  25.0  20.0  27.5                                            Ag 0.15     45.0  40.0  60.0  48.3                                            SPG 0.1 + Ag 0.15                                                                         25.0  12.5  12.5  16.7                                            SPG 0.1 + Ag 0.15                                                                         5.0   0.0   0.0   1.7                                             SPG 10.0 + Ag 0.15                                                                        5.0   10.0  2.5   5.8                                             SPG 20.0 + Ag 0.15                                                                        20.0  25.0  12.5  19.2                                            __________________________________________________________________________     Note 1. SPG: SPGM                                                             Note 2. Ag: Antigen                                                      

EXAMPLE 5

The same operation was conducted as in Example 4 except thatscleroglucan (hereinafter referred to simply as SG) instead of SPG-M.

The results thus obtained are shown in Table 5. It is evident from Table5 that when SG and vaccine are used in combination, the mortality isreduced as compared with the single use of vaccine even though the doseof the vaccine is reduced to one third. The ratio of antigen to SG ispreferably from 0.015 to 0.15.

                                      TABLE 5                                     __________________________________________________________________________    Enhancement of efficacy of orally administered vaccine by SG                  Contents of SG and Ag                                                                     Mortality (%)                                                     in diet (g/kg)                                                                            Repricate 1                                                                         Repricate 2                                                                         Repricate 3                                                                         Pooled Mortality (%)                            __________________________________________________________________________    Control     57.5  45.0  62.0  54.8                                            Ag 0.45     40.0  25.0  20.0  28.3                                            Ag 0.15     50.0  40.0  60.0  50.0                                            SG 0.1 + Ag 0.15                                                                          25.0  17.5  12.0  18.2                                            SG 0.1 + Ag 0.15                                                                          2.5   5.0   0.0   2.5                                             SG 10.0 + Ag 0.15                                                                         5.0   10.0  5.0   6.7                                             SG 20.0 + Ag 0.15                                                                         12.5  20.0  20.0  17.5                                            __________________________________________________________________________     Note 1. SG: Scleroglucan                                                      Note 2. Ag: Antigen                                                      

EXAMPLE 6

SPG-P or SG was injected i.p. twice to a salmon at the rate of 20 mg/kgevery two days. In two days after the last injection, the pronephoros ofthe salmon was excised and the living cell suspension of the phagocyte(about 1×10⁷ cells/ml) was prepared. A killing baker's yeast suspensionhaving about 2-2.5 times the cell number of the phagocyte was mixed tothe suspension of the phagocyte, and incubated at 17° C. for one hour.Then, a part of the suspension mixture was smeared on a slide glass, anddried and dyed with a Wright solution, followed by observation by meansof a microscope.

The phagocytic index (PI) was calculated in accordance with thefollowing formula: ##EQU1##

As control, saline was administered instead of the glucans to salmon andPI was calculated in the same manner as above.

The results are shown in Table 6. It is evident from Table 6 that the PIvalues for SPG-P and SG are high as compared with control, such showingstimulation of the non-specific immune system.

                  TABLE 6                                                         ______________________________________                                        Change in phagocytic activity of salmon's                                     pronephoros cells by SPG-P and SG                                                    Number of fish tested                                                                      Phagocytic Index (PI)                                     ______________________________________                                        Control  10              2.35 ± 0.75 (a)                                   SPG-P    10             10.53 ± 3.24 (b)                                   SG       10             12.51 ± 4.72 (b)                                   ______________________________________                                    

EXAMPLE 7

SPG-M or SG was mixed to a diet to have the composition as shown inTable 7 (i.e. 1 g/kg diet). Then, the diet was orally administered tosalmon in an amount of 2% of the weight for 7 days. In two days afterthe administration, the pronephoros of the salmon was excised and PI wascalculated in the same manner as in Example 6. As control, a dietcontaining no SPG-M or SG was used.

The results thus obtained are shown in Table 8. It is evident from Table8 that the PI values for SPG-M and SG are high as compared with control,such showing stimulation of the non-specific immune system.

                  TABLE 7                                                         ______________________________________                                        Composition for diet                                                          Composition         Amount (g/kg diet)                                        ______________________________________                                        Steam Dried Herring Meal                                                                          558.66                                                    Dried Whey           84.6                                                     Blood Flour          48.17                                                    Euphasids (whole frozen)                                                                           21.02                                                    Wheat Middling      126.19                                                    Vitamin Supplement   2.41                                                     Mineral Supplement   7.27                                                     Choline Chloride (60%)                                                                             4.60                                                     Ascorbic Acid        1.84                                                     Permapell            13.81                                                    Herring Oil          91.38                                                    SPG-M/SG             1.00 (0) *                                               α-Cellulose    39.05 (40.05) *                                          ______________________________________                                         * The value in the parenthesis is for control.                           

                  TABLE 8                                                         ______________________________________                                        Change in phagocytic activity of salmon's                                     pronephoros cells by SPG-M and SG                                                    Number of fish tested                                                                      Phagocytic Index (PI)                                     ______________________________________                                        Control  10             2.81 ± 0.95 (a)                                    SPG-M    10             9.53 ± 3.36 (b)                                    SG       10             10.36 ± 3.47 (b)                                   ______________________________________                                    

Thus the results show that β-1,3-glucans are effective both as means ofstimulating the immune system of fish and as means of improving theefficacy of vaccines.

The results show effect against A. salmonicida, the causative agent infurunculosis for which published vaccination attempts have yielded poorresults. But the same effect could, it is believed, be achieved againstbacterial kidney disease (BKD) caused by the organism Renibacteriumsalmoninarum which is also a pathogen of salmonid fish.

The above experimental results are directed to fish belonging to thefamily salmonidae. However, the experimental results are equallyapplicable to other species, for example ornamental fishes, pet, hobbyfishes, carp, sea bream and the like. In particular there is nothingthat would lead the skilled worker to believe that the compositions arenot effective for all fish but the invention is of particular interestin the treatment of aquaculture fish. There is a substantial homogeneityin the immune systems of all fish species.

The embodiments of the inventnion in whcih an exclusive property orprivilege is claimed are defined as follows:
 1. A method to stimulatethe immune system of fish that comprises administering to the fish aβ-1,3-glucan having a β-1,3-linked main chain with β-1,6-linked singleglucose side chains.
 2. A method as claimed in claim 1 in which the fishis a species belonging to the family salmonidae.
 3. A method as claimedin claim 1 in which the glucan is scleroglucan (SG).
 4. A method asclaimed in claim 1 in which the glucan is schizophyllan (SPG).
 5. Acomposition to enhance the efficacy of a fish vaccine comprising anantigen source and a β-1,3-glucan having a β-1,3-linked main chain withβ-1,6-linked single glucose side chains with an antigensource/β-1,3-glucan weight ratio of from 0.015 to 0.15.
 6. A compositionas claimed in claim 5 in which the antigen source is a vaccine.
 7. Acomposition as claimed in claim 5 in which the glucan is selected fromthe group consisting of schizophyllan (SPG) and scleroglucan (SG).
 8. Anagent to administer to a fish to stimulate the immune system of thefish, comprising an effective amount of β-1,3-glucan having aβ-1,3-linked main chain with β-1,6-linked single glucose side chains andfish food as a carrier to enable administration to the fish by mouth.